Primary open-angle glaucoma (POAG) is a leading cause of blindness worldwide. A primary risk factor for the development and progression of POAG is elevation of intraocular pressure (IOP), caused by an increase in aqueous outflow resistance. Most of this resistance is believed to be generated in the juxtacanalicular connective tissue (JCT) and modulated by the inner wall endothelium of Schlemm?s canal (SC), and its giant vacuoles and pores. However, the exact mechanisms that regulate outflow resistance remain unclear. Our long-term goal is to understand the mechanisms that regulate aqueous outflow resistance in normal eyes and how this resistance is increased in POAG. In our last funding period, we developed global imaging, a technique that can visualize the outflow pattern around the circumference of the eye and distinguish areas of high, low, or non-flow in the trabecular meshwork (TM), SC, and the distal episcleral veins. In these three parts, we found aqueous outflow to be segmental. We defined the area with active flow as the effective filtration area (EFA). We found inverse relationships between EFA and both IOP and outflow resistance. We also found that EFA and outflow facility increased in eyes treated with methods of lowering IOP: Rho-kinase inhibitors, gene modification, and minimally invasive glaucoma surgery (MIGS) using TM bypass devices. Based on these results, our goal of this project is to determine what mechanisms contribute to the regulation of EFA. We will distinguish morphological features of high, low, and non-flow areas, and determine whether we can increase EFA to lower IOP by converting non/low-flow areas to high-flow areas. To achieve our objectives, we developed a 3D electron microscopy method to reliably provide volumetric and geometric quantitation of giant vacuoles, pores, and cellular connections between the SC inner wall and its underlying JCT. We will test our hypothesis that cellular connections in the inner wall endothelium modulate giant vacuole and pore formation, thereby regulating EFA. We will also pioneer a novel 3D cell culture device with real-time imaging to scrutinize changes in cytoskeletal structure and giant vacuole formation after Rho-kinase inhibitor treatment. Importantly, we have enhanced the global imaging technique by combining it with fluorescein angiography to distinguish flow patterns before and after an IOP-reducing treatment. This offers the opportunity to identify newly converted high-flow areas arising from use of Rho-kinase inhibitors. These innovative methods allow us to address the clinical debate as to whether MIGS devices should be placed in high or non-flow areas to optimize post-operative IOP reduction. Our Specific Aims are: 1. To differentiate structural changes along inner wall of SC in high-flow areas compared to low/non- flow areas of normal and POAG eyes; 2. To determine effect of Rho-kinase inhibitors on giant vacuoles and pore formation; 3. To determine the best location (high, low adjacent to high, or non-flow area) to place a TM bypass stent to effectively increase EFA. The results of this study will advance our understanding of how EFA and outflow resistance are regulated, and potentially improve current clinical methods of IOP reduction in POAG.